Toll-like receptors (TLRs) are a large family of type I transmembrane proteins that function as “pattern recognition receptors.” These receptors constitute an integral component of the innate immune system as they are able to recognize microbial products and pathogen-associated molecular patterns (PAMPs), as well as endogenous ligands associated with inflammation or danger-associated molecular patterns (DAMPs). TLRs thus sense a variety of conserved molecules. There are at least 13 recognized TLRs, termed TLR1, TLR2, TLR3, etc. Upon ligand binding, the receptors generally homodimerize, although TLR2 forms heterodimers with TLR1 or TLR6, with each dimer having a different ligand specificity. Activated TLRs initiate the innate immune response that enables the host to combat infection.
Each of the germline-encoded human TLRs is structurally similar, consisting of a leucine-rich repeat-containing extracellular domain, a membrane spanning helix and a cytosolic TIR (Toll/IL-1 Receptor) domain (2, 3). The TIR domain is a highly conserved structure which is found in both prokaryotic and eukaryotic species (4-6). Structurally, TIR domains consist of alternating β strands and a helices (7-9). Upon recognition of either a PAMP or DAMP ligand, the ectodomains of stimulated TLRs form an M-shaped structure in which the C-termini of ectodomains converge, leading to dimerization of cytosolic TIR domains (7, 10-12). TIR dimerization exposes a composite binding site which provides a surface for recruitment of downstream TIR-domain containing adapter molecules present in the cytoplasm that, in turn, trigger signal propagation. Complementary surface areas of TIR domains, called interfaces, are key for the signal-initiated mutual recognition of TIR-containing proteins and the assembly of functional TLR signaling complexes through TIR:TIR domain interaction. TIR domains do not have a common TIR-binding motif and interact via structurally distinct regions.
Adapter protein recruitment to activated TLRs is exceptionally important as it governs the specificity of the TLR response (13). There are four recognized adapter proteins. Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), also known as MyD88-adapter-like (Mal), is a TIR domain-containing adapter utilized by both TLR2 and TLR4 to “bridge” MyD88 (a second adapter protein) to the receptors and, in turn, activate NF-κB (14-17). In the case of TLR4, there are two signaling pathways: MyD88-dependent and independent. The MyD88-dependent pathway utilizes TIRAP to bridge TLR4 and MyD88. The MyD88-independent pathway uses a third adapter protein, TRAM (TIR domain-containing adapter-inducing interferon-β- (TRIF-) related adapter molecule), which bridges TRIF (the fourth adapter protein) and allows for activation of IRF3. TIRAP appears to share a binding site with TRAM located at the TLR4 TIR homodimer (10, 11).
TIR domains, whether in the TLRs or the adapter proteins, mediate transient interactions of signaling proteins involved in inflammatory signaling and host defense. TIR domains tend to interact with other TIR domains, yet functional TIR:TIR interactions are specific, as exemplified by the recruitment of specific TIR-containing adapter proteins in response to activation of a particular receptor. Some TIR-containing adapter proteins participate in multiple signaling pathways, while others interact with a smaller set of proteins. For example, MyD88 is a necessary adapter for all members of the IL-1R family and all TLRs with the exception of TLR3, whereas TIRAP participates only in TLR2 and TLR4 signaling. Despite considerable effort, the molecular mechanisms that determine specificity of TIR:TIR interactions are not understood.
Small size (3-3.5 nm) and generally globular shape of TIR domains implies that any binary TIR:TIR complex has a limited interface size, typically less than 900 Å, and therefore a binary TIR complex is relatively weak and unstable. The stability of receptor complexes is achieved through a simultaneous, cooperative interaction of several (more than two) TIR domains, and multiple interface sites are expected in each TIR domain involved in signaling. Although the general mechanism of TLR signaling complex assembly is now commonly accepted, the architecture of TLR signaling complexes and the specific interface sites within TIR domains that mediate proteins interactions required for signaling complex assembly are not well understood.
Control of innate immunity in response to infection is vitally important as an impaired response increases susceptibility to infection, while an uncontrolled response can lead to inflammatory disease (2). TLRs and adapter proteins are important therapeutic targets because excessive TLR signaling is a pathogenic mechanism in many inflammatory diseases, including sepsis.